Aging of the population and popularity of refractive surgery threaten adequacy of the cornea supply used for transplantation in the USA. World-wide, cornea donations already fall short, leaving 6-8 million individuals suffering corneal blindness. Engineered corneas, grown in the laboratory and available on demand, will ultimately help alleviate this public health problem. The long-term goal of this project is to develop corneal equivalents for transplantation using cultured human stem cells. Initially the project will focus on the corneal stroma, the connective tissue that makes up 90% of the corneal mass. We have recently discovered a population of cells from human corneal stroma with properties of adult stem cells. Unlike keratocytes, the human corneal stromal stem cells (hCSSC) can be passaged numerous times in culture but still retain the ability to become keratocytes in vivo and in vitro. We hypothesize that the human corneal stromal stem cells participate in response to corneal injury and can regenerate transparent stromal tissue both in vivo and in vitro.. This hypothesis will be tested (1) by demonstrating the influx of hCSSC into pathological human corneas and by documenting the fate of hCSSC introduced into normal and healing mouse corneas in vivo. (2) Lumican null mice which develop opacity similar to corneal scars and corneas scarred by injection of transforming growth factor beta will be treated with hCSSC to investigate observed ability of these cells to restore corneal transparency. (3) Tissue equivalents produced in vitro by hCSSC will be implanted into mouse cornea to investigate the molecular and structural requirements for transparency in stromal tissue. Successful accomplishment of these aims will elucidate biological roles of the newly discovered adult stromal stem cells and provide novel information about how the stromal ultrastructure and corneal transparency are maintained in vivo. The experiments will also develop essential technologies for cell-based therapy for corneal scars and for fabrication of bioengineered tissue which could be used directly for lamellar keratoplasty or serve as the basis of a fully bioengineered cornea.